CN113773292B

CN113773292B - Washing-free AIEgen fluorescent probe targeting lipid droplets and preparation method and application thereof

Info

Publication number: CN113773292B
Application number: CN202111158091.8A
Authority: CN
Inventors: 胡磊; 王慧; 沈学彬; 于坤; 沈舒婷; 王亚轩
Original assignee: Wannan Medical College
Current assignee: Wannan Medical College
Priority date: 2021-09-29
Filing date: 2021-09-29
Publication date: 2022-06-10
Anticipated expiration: 2041-09-29
Also published as: CN113773292A

Abstract

The invention discloses a washing-free AIEgen fluorescent probe targeting lipid droplets and a preparation method and application thereof, wherein coumarin and diphenylamine structural units with better lipid solubility and stronger electron donating capability are introduced into the structure of the washing-free AIEgen fluorescent probe targeting lipid droplets to prepare a coumarin derivative, which has obvious AIE property and larger Stokes displacement, can quickly target lipid droplet positions in cells under the washing-free condition, and can dynamically monitor the migration process of lipid droplets in the cells and the quantity change of the lipid droplets in the cells in real time.

Description

Washing-free AIEgen fluorescent probe targeting lipid droplets and preparation method and application thereof

Technical Field

The invention relates to a washing-free AIEgen fluorescent probe targeting lipid droplets and a preparation method and application thereof, in particular to an AIEgen fluorescent probe with aggregation-induced emission property, low biotoxicity and targeting lipid droplets and a preparation method and application thereof.

Background

Lipid droplets are not only important energy storage sites in cells, but also complex, motile, dynamically changing multifunctional subcellular organelles that participate in many important cellular processes including lipid metabolism, membrane transport, cell activation and apoptosis, and protein degradation. Abnormalities in lipid droplet levels are closely related to many diseases, such as obesity, type II diabetes, fatty liver, cardiovascular disease, cancer, neutral lipid storage disease and Niemann Pick C disease. According to literature, the higher number of lipid droplets in cancer cells than in normal cells is reported because cancer cells require more energy provided by lipid droplets to accelerate cell proliferation. Therefore, the number of lipid droplets can be used as a potential tumor diagnosis index.

The current methods for detecting lipid droplets include an immune tissue method, a nuclear magnetic method, a Raman scattering method and a fluorescence imaging method. Fluorescence imaging methods are becoming more and more popular with many researchers because of their advantages such as ease of operation, high sensitivity, and ease of direct observation, as compared to other methods. Therefore, the real-time and in-situ monitoring of the change of the number of lipid droplets in organisms by using the fluorescent probe plays an important role in revealing the occurrence and the development of lipid droplet related diseases. The lipid drop dyes which are commercialized at present comprise HCS LipidTOX series, BODIPY series and the like, but in the practical use process, the commercialized dyes have the disadvantages of long incubation time, small Stokes shift, complicated cleaning process, aggregation-induced fluorescence quenching (ACQ) and the like.

In recent years, a newly emerging aggregation-induced emission (AIE) material can well solve the ACQ problem, and the AIE material has been widely used in the fields of biosensing, bioimaging, biomedicine, and the like. However, the structure and synthesis route of the existing aggregation-induced emission (AIE) material are complex. Therefore, it is still challenging to develop a wash-free lipid droplet targeted fluorescent probe with large stokes shift that has AIE properties, simple structure, and convenient synthesis.

Disclosure of Invention

In order to solve the technical problems, the invention provides a wash-free AIEgen fluorescent probe targeting lipid droplets, a preparation method and an application thereof, the wash-free AIEgen fluorescent probe has obvious AIE properties and larger Stokes shift, can quickly target lipid droplet positions in cells under the wash-free condition, and can dynamically monitor the migration process of lipid droplets in the cells and the change of the number of the lipid droplets in the cells in real time.

The technical scheme adopted by the invention is as follows:

a washing-free AIEgen fluorescent probe targeting lipid droplets, wherein the structural formula of the AIEgen fluorescent probe is as follows:

the invention also provides a preparation method of the washing-free AIEgen fluorescent probe targeting lipid droplets, which comprises the following steps: dissolving a compound M in a solvent, then sequentially adding ethyl acetoacetate and piperidine, carrying out reflux reaction for 5-5.5h, and then concentrating, filtering and recrystallizing to prepare the AIEgen fluorescent probe of the wash-free targeted lipid drop;

the structural formula of the compound M is as follows:

the molar ratio of the compound M to the ethyl acetoacetate to the piperidine is 1: 1.5-1.6: 0.6-0.8.

The solvent is absolute ethyl alcohol, and the concentration of the compound M in the absolute ethyl alcohol is 0.05-0.1 mmol/mL.

The recrystallization is carried out by using absolute ethyl alcohol.

The invention also provides application of the washing-free AIEgen fluorescent probe targeting lipid droplets in positioning lipid droplets in cells, and the washing-free AIEgen fluorescent probe can target lipid droplet positions in cells within 1 min.

The invention also provides application of the wash-free AIEgen fluorescent probe targeting lipid droplets in the fusion and migration process of lipid droplets in cells.

The invention also provides application of the wash-free AIEgen fluorescent probe targeting lipid droplets in monitoring the change of the number of lipid droplets in cells.

The invention provides a washing-free AIEgen fluorescent probe targeting lipid droplets, which is characterized in that a coumarin and diphenylamine structural unit with better fat solubility and stronger electron donating capability is introduced into the structure of the washing-free AIEgen fluorescent probe, so that a coumarin derivative is prepared, the coumarin derivative has obvious AIE property and larger Stokes displacement, and the washing-free AIEgen fluorescent probe targeting lipid droplets can be used as the washing-free AIEgen fluorescent probe targeting lipid droplets, quickly target lipid droplet positions in cells under the washing-free condition, and dynamically monitor the migration process of the lipid droplets in the cells and the quantity change of the lipid droplets in the cells in real time.

The invention also researches the fluorescence behavior of the AIEgen fluorescent probe of the wash-free targeted lipid drop in the lipid drop environment in vitro, and the result shows that the AIEgen fluorescent probe of the wash-free targeted lipid drop shows obvious fluorescence enhancement after being acted with the O/W emulsion. The AIEgen fluorescent probe of the visible wash-free targeted lipid droplet has important research value when being used as a wash-free lipid droplet specific targeted fluorescent probe for monitoring the dynamic change of the lipid droplet in the cell.

Compared with the prior art, the invention has the following advantages:

1. the structure and the preparation method of the washing-free AIEgen fluorescent probe targeting lipid droplets are simple, the reaction conditions in the preparation process are mild, the post-treatment is simple, and the raw materials are simple and easy to obtain.

2. The washing-free lipid drop targeted AIEgen fluorescent probe provided by the invention can be quickly positioned at a lipid drop position in a cell, and can monitor the fusion and migration processes of the lipid drop in real time.

3. The washing-free AIEgen fluorescent probe targeting lipid droplets can monitor the change of the number of lipid droplets in cells.

4. Compared with a commercial lipid drop probe, the wash-free lipid drop targeting AIEgen fluorescent probe provided by the invention has larger Stokes shift, the excitation wavelength is 442nm, the emission wavelength is 578nm, and the Stokes shift is 136nm, so that the background interference can be reduced.

5. After the washing-free AIEgen fluorescent probe targeting lipid droplets provided by the invention acts with cells, the washing-free AIEgen fluorescent probe can be used for development test without washing.

Drawings

FIG. 1 is a structural formula of a wash-free AIEgen fluorescent probe targeting lipid droplets;

FIG. 2 is an infrared spectrum of a leave-on lipid droplet targeted AIEgen fluorescent probe;

FIG. 3 is a nuclear magnetic diagram of the hydrogen spectrum of the AIEgen fluorescent probe of the wash-free targeting lipid droplet;

FIG. 4 is a carbon spectrum nuclear magnetic map of the AIEgen fluorescent probe for the wash-free targeted lipid droplet;

FIG. 5 shows fluorescence emission spectra of compound L in mixed solutions of ethanol/water with different volume fractions. The inset is a fluorescent photograph of compound L in absolute ethanol (water content 0%) and 1% by volume ethanol aqueous solution (water content 99%) under 365nm ultraviolet lamp illumination;

FIG. 6 shows confocal microscopy imaging of compound L after interaction with HepG2 cells;

figure 7 is the co-localization results of compound L with different commercial dyes (including lipid droplet commercial, mitochondrial and lysosomal commercial dyes): FIGS. A1-A4 Compound L development; FIGS. B1-B4 correspond to the development results for different commercial dyes, respectively; FIGS. C1-C4 correspond to the superimposed results of FIGS. A1-A4 and B1-B4, respectively; plots D1-D4 correspond to Pearson's coefficient values, respectively, for the co-localization results;

FIG. 8 is an image of the cells taken at 1min intervals after 20min of co-culture of Compound L with HepG2 cells;

FIG. 9 is an image A of cells after Hela cells were stimulated with different concentrations of oleic acid for 6h, and then compound L was added; stimulating Hela cells with 100 mu M oleic acid for different times, and adding compound L to form a cell image B;

FIG. 10 is a development of Compound L at various time points of action with cells;

FIG. 11 is a plot of fluorescence emission spectra of compound L after interaction with lipid droplet environment and other analytes, including O/W emulsion, liposomes, ADP, ATP, BSA, Cys, DNA, GSH, RNA, proline, serine, threonine, valine, and histone.

FIG. 12 is a hydrogen spectrum nuclear magnetic diagram of Compound LC2 in comparative example 1;

FIG. 13 is a carbon spectrum nuclear magnetic diagram of Compound LC2 in comparative example 1;

fig. 14 shows fluorescence emission spectra of compound LC2 in comparative example 1 in different volume fractions of ethanol/water mixed solution. The inset is a fluorescent photograph of compound L in absolute ethanol (water content 0%) and 20% by volume aqueous ethanol (water content 80%) under 365nm uv illumination;

FIG. 15 shows the results of confocal microscopy of compound LC2 of comparative example 1 after interaction with HepG2 cells;

fig. 16 is a development of compound LC2 in comparative example 1 at different time points in response to the cells.

Detailed Description

The present invention will be described in detail with reference to examples.

Example 1

A washing-free AIEgen fluorescent probe targeting lipid droplets has a structural formula as follows:

the preparation method and the synthetic route of the washing-free AIEgen fluorescent probe targeting lipid droplets are as follows:

after compound M (0.43g,1.5mmol) was added to a 100mL round-bottomed flask and completely dissolved in 20mL ethanol, ethyl acetoacetate (0.29g,2.25mmol) and 100. mu.L piperidine were added in this order, and after 5 hours of reflux reaction, most of the solvent was evaporated under reduced pressure, and then cooled to room temperature, a solid precipitated, which was filtered under reduced pressure, and the crude product was recrystallized from ethanol to give 0.37g of compound L as a yellow solid in 69% yield. The infrared, hydrogen and carbon spectra are shown in FIGS. 2, 3 and 4.

IR(KBr,cm^-1)selected bands:3063,2362,1718,1675,1614,1579,1491,1432, 1358,1348,1338,1288,1276,1215,1202,1135,1074,977,856,753,694,640,504.

¹H NMR(600MHz,d₆-DMSO)δ:8.54(s,1H),7.73(d,J＝8.8Hz,1H),7.48 (t,J＝7.8Hz,4H),7.31(m,6H),6.72(m,1H),6.46(d,J＝1.7Hz,1H),2.53(s,3H).

¹³C NMR(151MHz,d₆-DMSO)δ 194.97,169.68,159.50,157.30,153.90, 147.77,145.19,132.55,130.70,127.41,126.89,119.16,115.36,111.29,102.83, 69.14,30.55,16.92.

ESI-MS:356.1281([M+1]⁺).

When the compound L was dispersed in ethanol/water mixed solutions of different volume fractions and fluorescence emission spectra at an excitation wavelength of 442nm were measured, as shown in fig. 5, it can be seen from the graph that in an ethanol aqueous solution with a water content of 99% (ethanol aqueous solution with a volume fraction of 1%), the compound L had a strong fluorescence emission intensity at 578nm, and thus, the compound L had significant AIE properties.

Example 2

Biological study of Wash-free lipid droplet-Targeted AIEgen fluorescent probes

1. Lipid droplet targeting study

The compound L prepared in example 1 was dissolved in dimethyl sulfoxide to a concentration of 10^-2M, diluting the mother liquor to 10 mu M with the culture medium for later use. Passable human hepatoma cells (HepG2) were seeded on a confocal laser-induced cell dish (NEST cat # 801002) at 5X 10 cells per well⁵The cells can be used when the cell density in the dish is increased to 50%. The culture medium in the dish was removed first, and 1mL of the above-mentioned compound L solution to be used was added thereto in a solution containing 95% air and 5% CO₂The gas incubator was operated at 37 ℃ for 20 minutes, and the following two sets of experiments were set up: (1) washing with PBS buffer solution for 2 times; (2) washing was not performed with PBS buffer solution. Observing the result on a Leica TCS SP8 laser confocal microscope device, and setting the excitation wavelength to be 442 nm; as shown in fig. 5, it can be seen that after the compound L and the cells are stained, the washing is substantially consistent with the imaging result of the cells without washing, which indicates that after the compound L and the cells are acted, the compound L can be used for developing the test compound L without washing, and can be targeted to the lipid droplet position in the cells without washing.

In order to prove that the compound of the invention can target lipid drop sites in cells, four groups of human liver cancer cells (HepG2 cells) and 1mL of 10 μ M compound L are acted for 20min at 37 ℃ according to the method for culturing the cells, washed for 2 times by PBS buffer solution, and then subjected to laser confocal development at an excitation wavelength of 442nm, wherein the experimental result is shown in FIG. 6;

then co-cultured with commercial dyes BODIPY 493/503 (lipid droplet commercial stain), HCS LipidOX Deep Red (lipid droplet commercial stain), Mitotracker Red (mitochondrial commercial stain) and Lysotracker Red (lysosomal commercial stain) for 20min, washed 2 times with PBS buffer solution, and laser confocal development was performed at excitation wavelengths of 488nm, 633nm, 579nm and 577nm, respectively, and the development results are shown as B1-B4 in FIG. 7; FIGS. C1-C4 correspond to the superimposed results of FIGS. A1-A4 and B1-B4, respectively; plots D1-D4 correspond to Pearson coefficient values for co-localization results, respectively. From fig. 3, it can be demonstrated that compound L is able to target lipid droplet sites within cells.

2. Study on lipid droplet fusion and migration process

According to the experimental method in the lipid droplet targeting study, the cells were cultured, human hepatoma cells (HepG2 cells) were cultured with 1mL of 10. mu.M compound L at 37 ℃ for 20min, washed with PBS buffer solution for 2 times, and then fresh medium was added, and laser confocal imaging was performed at an excitation wavelength of 442nm, as shown in FIG. 8, it can be seen that the compound L can monitor the process of fusion and migration of lipid droplets in real time.

3. Monitoring changes in the number of intracellular lipid droplets

Oleic acid induced lipid droplet production by cells, and the number of lipid droplets in human cervical cancer (Hela) cells was lower than that in human hepatoma cells, so that the change in lipid droplets stimulated by oleic acid was monitored using compound L, as a model of Hela cells. Two sets of experiments were set up as follows: (1) dividing Hela cells into four groups, adding 1mL of oleic acid (0, 25, 50 and 100 mu M) with different concentrations and Hela cells into a dish for co-culture for 6h, removing the culture medium in the dish, adding 1mL of fresh culture medium (DMEM high-sugar medium containing 10% of serum and 1% of double antibody) containing 10 mu M of compound L, continuing to culture for 20min, and washing for 2 times by using PBS buffer solution; (2) dividing the Hela cells into four groups, adding 1mL of oleic acid with the concentration of 100 mu M into the dishes, culturing the Hela cells for different times with the Hela cells, wherein the culturing time is 0, 2, 4 and 6 hours, removing the culture medium in the dishes, adding 1mL of fresh culture medium (DMEM high-sugar medium containing 10% of serum and 1% of double antibody) containing 10 mu M of compound L, continuing culturing for 20min, and washing for 2 times by using PBS buffer solution; confocal laser imaging was performed at an excitation wavelength of 442nm, as shown in FIG. 9. As can be seen from A and B in FIG. 9, the number of intracellular lipid droplets is increasing and the fluorescence intensity is increasing, both when cells are stimulated with oleic acid at different concentrations and when cells are stimulated with oleic acid at the same concentration for different times, indicating that compound L can be used to monitor the change in the number of intracellular lipid droplets.

4. Time to lipid droplet study

Human hepatoma cells (HepG2 cell) were seeded in confocal dishes and used when the cell density increased to 50%. HepG2 cells are placed under a laser confocal microscope, the excitation wavelength is set to be 442nm, areas are selected for shooting, 1mL of 10 mu M compound L is added, shooting is carried out at intervals of 1min, and the experimental result is shown in figure 10.

5. Research on recognition mechanism

The fluorescence behavior of compound L in the presence of lipid droplet environment and other analytes was examined using fluorescence emission spectroscopy. The O/W emulsion is selected to simulate lipid drop environment, and other analytes are selected from liposome, Adenosine Diphosphate (ADP), Adenosine Triphosphate (ATP), Bovine Serum Albumin (BSA), cysteine (Cys), calf thymus DNA, Glutathione (GSH), ribonucleic acid (RNA), proline, serine, threonine, valine and histone. Preparing an O/W emulsion: 63.9mg of glycerol trioleate, 0.0025mM of cetyltrimethylammonium bromide and 50mL of PBS buffer solution are added into a 100mL round-bottom flask in sequence and stirred vigorously at room temperature for 6 hours for standby. Preparing the liposome: 75.8mg of liposome was weighed, dissolved in 20mL of chloroform, the solvent was removed under reduced pressure at 37 ℃ and cooled to room temperature, 50mL of PBS buffer was added and the mixture was vigorously stirred at room temperature for 2 hours. The concentration of the mother liquor of ADP, ATP, BSA, DNA, RNA and histone is 5mg/mL, and the concentration of the mother liquor of Cys, GSH, proline, serine, threonine and valine is 10^-2mol/L. The mother liquor concentration of the compound L was 10^-3mol/L, and the solvent is dimethyl sulfoxide. mu.L of the mother solution of compound L was pipetted with a pipette and placed in 14 5mL volumetric flasks, No. 1 flask was used as a control, and the volume was adjusted to 5mL with PBS buffer, and No. 2 and No. 3 flasks were adjusted to 5mL with liposome and O/W emulsion, respectively, and 200. mu.L of the above-mentioned analyte was added to the remaining flasks and adjusted to 5mL with PBS buffer. The fluorescence intensity of the compound L in the O/W emulsion is higher than that of other analytes by setting instrument parameters to be 442nm, the width of a slit to be 5nm and the voltage to be 500V, and the experimental result is shown in figure 11.

Comparative example 1

Compound LC2, having the structural formula:

the preparation method of the compound LC2 is as follows:

in a 100mL round bottom flask, compound M (0.43g,1.5mmol) was added and completely dissolved with 20mL ethanol, 4-butoxyphenylacetonitrile (0.57g,3.0mmol) and 100 μ L piperidine were sequentially added, and after refluxing for 24 hours, 10mL of 4mol/L hydrochloric acid solution was added to the reaction solution, and the reaction was continued for 24 hours, after completion of the reaction, cooled to room temperature, poured into 200mL of an ice-water mixture, extracted with dichloromethane 3 times, the organic layer was collected, dried over anhydrous magnesium sulfate overnight, most of the solvent was distilled off, and separated and purified by silica gel column chromatography (petroleum ether/ethyl acetate ═ 40/1) to obtain lc20.11g as a yellowish green solid, yield: 16 percent of

The hydrogen spectrum and the carbon spectrum of compound LC2 prepared in this comparative example are shown in fig. 12 and 13, respectively.

¹H NMR(600MHz,d₆-DMSO)δ:8.07(s,1H),7.73(m,1H),7.68-7.63(m,3H), 7.57(d,J＝8.6Hz,1H),7.42(t,J＝7.7Hz,4H),7.20(t,J＝7.7Hz,4H),6.99(t,J＝7.0Hz,2H),6.81(d,J＝12.0Hz,1H),6.63(s,1H),4.00(t,J＝12.0Hz,2H),1.76 -1.69(m,2H),1.45(m,2H),0.95(t,J＝7.5Hz,3H).

¹³C NMR(151MHz,d₆-DMSO)δ:167.42,160.24,159.16,154.40,150.69, 146.23,132.19,131.97,130.44,129.95,129.12,126.43,125.63,116.52,114.62, 113.35,105.67,67.57,65.48,31.19,30.48,19.19,19.11,14.14,13.99.

When compound LC2 was dispersed in ethanol/water mixed solution of different volume fractions and fluorescence emission spectra at excitation wavelength of 400nm were measured, as shown in FIG. 14, it can be seen that compound LC2 has strong fluorescence intensity at 505nm in ethanol aqueous solution with water content of 80% (20% ethanol aqueous solution by volume fraction), and it can be seen that compound LC2 has obvious AIE property.

Comparing the resultsThe compound LC2 prepared in example was dissolved in dimethyl sulfoxide to give a concentration of 10^-2M, diluting the mother liquor to 10 mu M with the culture medium for later use. Passable human hepatoma cells (HepG2) were seeded on a confocal laser-induced cell dish (NEST cat # 801002) at 5X 10 cells per well⁵The cells can be used when the cell density in the dish is increased to 50%. The culture medium in the dish was removed first, and 1mL of the above ready-to-use compound LC2 solution in a solution containing 95% air and 5% CO was added₂The gas incubator was operated at 37 ℃ for 20 minutes, and the following two sets of experiments were set up: (1) washing with PBS buffer solution for 2 times; (2) washing was not performed with PBS buffer solution. Observing the result on a Leica TCS SP8 laser confocal microscope device, and setting the excitation wavelength to be 405 nm; as shown in fig. 15, it can be seen that compound LC2 can be targeted to lipid droplet sites within cells. The imaging effect of cells which are not washed is not as good as that of cells which are washed, which indicates that after the compound LC2 acts on the cells, the cells need to be washed for imaging intracellular lipid droplets.

Human hepatoma cells (HepG2 cell) were seeded in confocal dishes and used when the cell density increased to 50%. HepG2 cells are placed under a laser confocal microscope, the excitation wavelength is set to be 405nm, areas are selected for shooting, then 1mL of 10 mu M compound LC2 is added, shooting is carried out at intervals of 10min, the experimental result is shown in figure 16, as can be seen from the figure, the compound LC2 can enter the targeted lipid drop position of the cells within 10min, the background noise of the areas outside the cells is high, and the compound LC2 is a non-washing-free fluorescent probe for the targeted lipid drop.

The above detailed description of a leave-on lipid droplet-targeted AIEgen fluorescent probe, its preparation and use with reference to the examples is illustrative and not restrictive, and several examples can be cited within the limits set forth, so that variations and modifications that do not depart from the general concept of the present invention are intended to be within the scope of the present invention.

Claims

1. The washing-free AIEgen fluorescent probe targeting lipid droplets is characterized in that the structural formula of the AIEgen fluorescent probe is as follows:

。

2. the method for preparing the wash-free lipid-droplet-targeted AIEgen fluorescent probe according to claim 1, wherein the preparation method comprises the following steps: dissolving a compound M in a solvent, then sequentially adding ethyl acetoacetate and piperidine, carrying out reflux reaction for 5-5.5h, and then concentrating, filtering and recrystallizing to prepare the AIEgen fluorescent probe of the wash-free targeted lipid drop;

the structural formula of the compound M is as follows:

。

3. the preparation method according to claim 2, wherein the molar ratio of the compound M to the acetoacetic acid ester to the piperidine is 1: 1.5-1.6: 0.6 to 0.8.

4. The method according to claim 2 or 3, wherein the solvent is absolute ethanol, and the concentration of compound M in absolute ethanol is 0.05 to 0.1 mmol/mL.

5. The production method according to claim 2 or 3, wherein the recrystallization is a recrystallization using anhydrous ethanol.